The activity of biological molecules is often greatly influenced by the presence of one or more asymmetric centers. For example, R-asparagine is a sweetener, while the S enantiomer tastes acidic; S-S ethanbutol is a tuberculostatic drug, yet the R-R form causes blindness. Typical organic syntheses yield racemic mixtures, which often require a cumbersome separation to obtain the desired enantiomer. So, for the synthesis of pharmaceuticals, fragrances, sweeteners, etc., direct stereoselective syntheses yielding only the desired chiral configurations are highly preferred over non-stereoselective routes requiring separation of racemic mixtures.
Nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) (collectively referred to as NAD(P), NAD(P)+ in the oxidized form, and NAD(P)H in the reduced form) serve as cofactors in many enzyme catalyzed oxidation-reduction reactions. A broad range of applications for NAD-dependant enzyme reaction exists, particularly using dehydrogenases: production of amino acids by reductive amination of α-keto carboxylic acids (L-leucine, L-alanine, L-vanilline, etc.); production of hydroxy-acids (for example, the conversion of pyruvate into L-lactate, and production of α-ketobutyrate into L-hydroxy acid); reduction of aldehydes and ketones; synthesis of alcohols that have applications in perfumes and food additives; selective hydroxylation and dehydrogenation of steroids (via hydroxylation of progesterone, and dehydrogenation of an intermediate in the synthesis of chenodeoxycholic acid, a gallstone drug); and a variety of environmental processes involving carbon dioxide transformation. Dehydrogenase-catalyzed syntheses would thus become interesting from an economic view point if the required NADH (and NADPH) cofactor was commonly available, or could be continuously regenerated from NAD+ (or NADP+) (see, for example, R. Devaux-Basseguy, A. Bergel, M. Comtat, Potential Applications of NAD(P)-dependent oxidoreductases in synthesis: a survey, Enzyme Microb. Technol., 1997, 20, 248-258).
Chemical, photo-chemical, enzymatic, biologic and electro-chemical methods to regenerate NADH and NADPH have been studied extensively (see, for example, H. K. Chenault, G. M. Whitesides, Regeneration of Nicotinamide Cofactors for use in Organic Synthesis, Applied Biochemistry and Biotechnology, 14,147-194 (1987)). From an electro-chemical point of view, one can regenerate NADH directly or indirectly. Direct conversion of NAD+ into the active NADH at the electrode (FIG. 1A) is difficult: intermediate radicals dimerize and various inactive isomers are formed. These problems can be overcome, but not without significant extra effort, including functionalization of NAD by precise pretreatment of the electrodes, or by using additional hydrogenase enzymes. In the indirect regeneration of NADH from NAD+, a mediator accepts electrons from the electrode and provides these to an enzyme that regenerates NADH from NAD+ (FIG. 1B).
Use of flavin adenine dinucleotide (FAD) as the mediator has been proposed because of its stability in electrochemical cycles. However, the oxidation of the desired NADH species by the FAD mediator is spontaneous at pH 7.0 without involvement of an enzyme:NADH+FAD+H+⇄NAD++FADH2ΔG′°=−20.3 kJ/mol  (1)
In classical batch reactors, FADH2 is not sufficiently stable and its concentration is never great enough in solution to shift the equilibrium in the reverse direction to generate NADH. Bergel et al. showed that shifting the equilibrium to favor the reverse reaction using a thin layer electrochemical cell is possible, yet difficult (A. Bergel and M. Comtat, “Thin-layer spectroelectrochemical study of the reversible reaction between nicotinamide adenine dinucleotide and flavin adenine dinucloeotide,” Journal of Electroanalytical Chemistry, vol. 302, 219-231 (1991)). Although this thin layer cell works well for analytical purposes, using this small, non-flowing design for production would be difficult, if not impossible.
Laminar flow electrochemical synthesis has been described (U.S. Pat. No. 6,607,655). This cell includes two parallel electrodes placed in close proximity (typically 0.25-1 mm) where a single stream of reactants is passed between the electrodes.
Patents involving microfluidic systems or methods involving laminar flow are also known. Published patent application 20030124509 offers a comprehensive overview of microfluidic devices in chemical systems; electrochemical systems are not described. U.S. Pat. No. 6,713,206 describes a galvanic electrochemical cell including multistream laminar flow. Graphite plates as current collectors have also been described (for example, published patent application 20010033958).